PROJECT SUMMARY Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG) is in the top tier of AMR l threats as defined by WHO. Over the past decades, NG has developed resistance to all antimicrobials previously recommended for treatment of gonorrhea, leaving dual therapy of ceftriaxone plus azithromycin as currently the only appropriate option for empirical first-line therapy in most countries world-wide. Now NG strains have been reported resistant to both ceftriaxone and azithromycin. Conversely, although ciprofloxacin is no longer recommended by the CDC for the treatment of NG, recent studies suggest that a large percentage of GC infections could be potentially treated with ciprofloxacin. With the continued evolution of AMR, there is an urgent need for personalized treatment approaches that target an individual infection, in contrast to the current ?globally uniform? empiric approach. However, this requires clinicians to know drug resistance or susceptibility quickly enough to inform prescription decisions. To mitigate the emergence and spread of AMR in NG, CDC periodically publishes STD treatment guidelines to assist clinicians. These guidelines are informed by susceptibility data generated by the national CDC Gonococcal Isolate Surveillance Project (GISP). GISP?s impact, however, is being jeopardized by technological evolution. Determining AMR requires prolonged (24-48 hours) microbiological cultivation in sophisticated laboratory facilities. But the advent of nucleic acid amplification tests (NAAT) with enhanced speed and accuracy has supplanted culture-based diagnosis of NG infections, leading to limited specimen collection for culture and loss of capability to perform culture of NG in most testing clinics. Consequently, the success of widespread NAAT has inadvertently created a critical void in AMR testing. We propose to develop a complete diagnostic solution capable of performing identification (ID) of NG infection and phenotypic antimicrobial susceptibility testing (AST). Specifically, ID is achieved using PCR to detect NG- specific DNA markers; while AST is carried out using quantitative PCR to measure the difference in nucleic acid markers (bacterial DNA or RNA) which correlate with the physiologic state of pathogen between drug- treated samples and no-drug controls. Our combined ID-AST platform, which capitalizes on innovative advances in NAAT and microfluidics, has the potential to deliver all essential NG diagnostic information specific to each suspected patient at the POC to tailor personalized treatment; its practical design is also well- suited to resolve the technical challenges confronting GISP for routine surveillance of NG AMR. We propose the following aims: 1) to develop a streamlined diagnostic protocol for integrated ID and AST of NG; 2) to develop a droplet microfluidic cartridge implementing the integrated ID-AST assay; 3) system integration and instrument development; and 4) analytical and clinical validation of the integrated system. To facilitate technology translation, a Product Development Plan for future clinical deployment is proposed.